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Many biological processes require a purification scheme to reduce the fermentation broth to its pure final product. Once citric acid is made in the production fermenter, the broth is still highly contaminated. There are many different types of purification methods (extraction, filtration, coagulation, etc.) but for citric acid recovery, the most commonly used method is precipitation with lime and sulfuric acid and filtration to produce free citric acid. The following tutoring will go through various purification techniques and then take you through a typical purification scheme for citric acid.
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Extraction
Extraction is used to liberate a product of microbial growth from the cells or cellular constituents that served as the enzyme source either by mechanical or non-mechanical means. Mechanical Extraction Mechanical disruption of the cell is easy to achieve on a small scale but can fail when used industrially High Pressure Homogenizer
- A positive displacement pump with an adjustable valve, has been used to break microorganisms like Aspergillus niger, Escherichia coli, and Bacillus megatherium.
When cell concentration is high, the spores or mycelia from the microorganism can clog the valve
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Extraction
Non-mechanical Extraction Desiccation
Solvent Extraction
- Liquid extraction of a product from soluble particles within the cell - Must choose solvent accordingly, and purification efforts will follow to recover product from solvent
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Precipitation
Precipitation is a procedure where the addition of a ionic solution to an ionic fermentation broth forms insoluble particles, where the desired product is usually contained within those particles. Ionic fermentation broths usually consist of enzymes or proteins. The ways to precipitate out a product can vary from simple pH and temperature changes to chemical reactions involving metal ions. Precipitation reactions are carried out in reactors, continuous and batch. Temperature and pH variation
Overall, most proteins and enzymes display increased solubility with increased temperature, but care must be taken to prevent loss of product By adjusting the pH, an enzymes polarity can be lowered so that it has a zero net charge; at this lowest polarity, the enzyme has low solubility in an aqueous solution
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Precipitation
Precipitation by Organic Solvents
By adding an organic solvent to an aqueous fermentation broth, the dielectric constant will decrease causing the solubility to decrease Often used industrially because its inexpensive and simple
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Centrifugation
Centrifugation involves separation of liquids and particles based on density. Centrifugation can be used to separate cells from a culture liquid, cell debris from a broth, and a group of precipitates. There are numerous types of centrifuges, but only a few will be presented here. Tubular Bowl Centrifuge
Most useful for solid-liquid separation with enzymatic isolation Can achieve excellent separation of microbial cells and animal, plant, and most microbial cell debris in solution
Centrifugation
Perforate Bowl Basket Centrifuge
Exception at separation of adsorbents, such as cellulose and agarose
Zonal Ultracentrifuge
Applied in the vaccine industry because it can easily remove cell debris from viruses Can collect fine protein precipitates Has been used experimentally to purify RNA polymerase and very fine debris in enzymes
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Filtration
Filters use a filter cloth or some porous material along with applied pressure to push smaller particles through the filter, thus separating elements of the solution based on size. Filtration for biological materials is generally completed using batch filtration, rotary drum filtration, or ultrafiltration methods. Batch Filtration
Usually performed under constant pressure with a pump that moves the broth or liquor through the filter Filter cake will build-up as filtration proceeds and resistance to broth flow will increase The filter press is the typical industrial version of a batch vacuum filter, using a plate and frame arrangement Can be used to remove cells, but does not work particularly well for animal cell debris or plant seed debris
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Filtration
Rotary Drum Filtration
Solution is vacuumed upward where it crosses a filter septum removed by a positive displacement pump Filter cake is removed after each rotation to give a fresh surface for filtration Rotary vacuum filters can be used to efficiently remove mycelia, cells, proteins, and enzymes, though a filter aid or precoat of the septum may be necessary
Ultrafiltration
Utilizes a membrane to separate particles that are much larger than the solvent used Successful removal occurs in the partical size range of 10 solvent molecular diameters to 0.5
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Filter precipitate
CST R
Filter
Ex. Plate filter, Rotary presses, rack-and-frame presses
To crack the calcium citrate precipitate, sulfuric acid is needed. The temperature of this reaction should stay below 60C. The reaction will produce free citric acid and a new precipitate, calcium sulfate, which will need to be removed later. The stoichiometric coefficients for this reaction are all one.
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CST R
In this filter, the calcium sulfate is washed away from the citric acid and the leftover biomass is removed. Again, the contaminants that were present in the fermentation broth can be removed by additional filtration means, such as microfiltration or ultrafiltration.
Filter
Ex. Plate filter, Rotary presses, rack-and-frame presses
2. Anhydrous
Processed to remove all water from end product Prepare by dehydrating the monohydrate citric acid product at a temperature above 36.6C
Kirk 16,17 KICgroup 1
Purification Conclusion
Once the product has been brought to the desired purity, it would be sent to packaging and distribution. In summary, there are many different methods and types of equipment that make up a purification scheme. Some of the more common types or purification were discussed in the first half of this tutorial. The second half of the tutorial dealt with a specific citric acid purification scheme that utilizes precipitation and filtration to recover free citric acid from a contaminated fermentation broth.
Purification
Citric Acid
References
KIC Chemicals. Citric Acid Monohydrate, USP (CoarseGranular). Retrieved April 21, 2004, from the World Web: http://www.kicgroup.com/citmonokc.htm Wide
Kirk, Raymond E., Othmer, Donald F. (Ed). (1949). Encyclopedia of Chemical Technology (Vol 4). New York City: Interscience Publishers, Inc. Wang, Daniel I. C., & Cooney, Charles L., Demain, Arnold L., Dunnill, Peter, Humphrey, Arthur E, Lilly, Malcolm D. (1979). Fermentation and Enzyme Technology. New York City: John Wiley & Sons.
Citric Acid